Immunoassay Methods Utilizing Trapping Conjugate

ABSTRACT

Methods are provided for determining the presence of a first ligand in a sample. In some embodiments depletion conjugates are used to deplete the ligands different from but related to the first ligands from the sample. In some embodiments, interim binding agents are used to enhance the test signal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/791,299, filed Oct. 23, 2017, which is a continuation of U.S.application Ser. No. 14/631,006, filed Feb. 25, 2015, now U.S. Pat. No.9,891,216, which claims benefit of U.S. Provisional Application Ser. No.61/974,060, filed Apr. 2, 2014, all of which are hereby incorporatedherein by reference in their entireties.

Related Patents

This application relates to co-owned U.S. Pat. Nos. 7,189,522,7,682,801, 7,879,597, 8,507,259, and 8,603,835 all of which are herebyincorporated by reference herein in their entireties.

BACKGROUND 1. Field

The subject disclosure relates broadly to immunoassay methods anddevices. More particularly, the subject disclosure relates to thedetection of one or more particular ligands in a body fluid possiblycontaining additional related ligands.

2. State of the Art

Many types of ligand-receptor assays have been used to detect thepresence of various substances, often generally called ligands, in bodyfluids such as blood, urine, or saliva. These assays involve antigenantibody reactions, synthetic conjugates comprising radioactive,enzymatic, fluorescent, or visually observable polystyrene or metal soltags, and specially designed reactor chambers. In all these assays,there is a receptor, e.g., an antibody, which is specific for theselected ligand or antigen, and a means for detecting the presence, andin some cases the amount, of the ligand-receptor reaction product. Sometests are designed to make a quantitative determination, but in manycircumstances all that is required is a positive/negative qualitativeindication. Examples of such qualitative assays include blood typing,most types of urinalysis, pregnancy tests, and AIDS tests. For thesetests, a visually observable indicator such as the presence ofagglutination or a color change is preferred.

Co-owned U.S. Pat. Nos. 7,189,522, 7,682,801, 7,879,597, and 8,507,259are directed to improved rapid detection assays utilizing a “dual path”lateral flow device. More particularly, the immunoassay device isprovided with a first sorbent strip that provides a first lateral orhorizontal flow path for a conjugate, and a second sorbent strip thatprovides a second lateral or horizontal flow path for a sample. A testsite having an immobilized ligand-binding mechanism is located on or inat least one of the strips, and the strips touch each other at the testsite. In use, the sample and a buffer solution are first provided to thesecond sorbent strip and flow over time to the test site along thesecond flow path (i.e., they do not immediately wet the test site). Ifthe sample contains ligand of interest, the ligand is captured at thetest site by the immobilized ligand-binding mechanism. Buffer solutionprovided to the first sorbent strip carries the conjugate to the testsite after the sample has reached the test site. If ligand is capturedat the test site, the conjugate binds to the captured ligand andprovides an indication of a “positive” test result; i.e., ligand ofinterest was present in the sample. If ligand is not captured at thetest site, the conjugate does not bind, and a “negative” test results isobtained; i.e., ligand of interest was not present in the sample. Acontrol line that captures conjugate may be provided near the test siteto confirm that the test was properly conducted. By providing separateflow paths for the sample and the conjugate, substantially highersensitivity and selectivity are obtained relative to standard lateralflow devices and reverse-flow devices utilizing single strips.

The dual path devices have also proved to be robust in providingaccurate sensitive results where the test site is provided with multipledifferent immobilized ligand-binding mechanisms; i.e., multiplexcapabilities. For example, separate test lines in a single DPP devicehave been provided for separately and accurately detecting HIV-1, HIV-2,and syphilis.

SUMMARY

In one embodiment a dual path immunoassay test cell device for detectingthe presence of a first ligand in a sample is provided with a firstsorbent material defining a first horizontal or lateral flow path and asecond sorbent material defining a second horizontal or lateral flowpath, the first and second sorbent materials overlying one another at atest site. The first flow path has a first location for receiving afirst solution, which, in the case of a liquid conjugate system is aconjugate solution, and which, in the case of a dry conjugate system isa buffer solution. Where a buffer solution is utilized, the firstsorbent material is provided with a first (mobile) conjugate locateddownstream of the first location. The second flow path has a secondlocation for receiving a second solution comprising a sample. In oneembodiment, the sample is a blood, urine, saliva, or other sample thatmay be mixed with buffer solution if desired, and immobilizedsecond-ligand binding molecules are located downstream of the secondlocation. The second-ligand binding molecules are related to the firstligand for which the sample is being tested but are not the same. Thesecond sorbent material is distinct or separate from the first sorbentmaterial. The test site is provided with first-ligand binding moleculessuch as immobilized antigens or antibodies or other molecules such asaptamers, nucleic acids, etc. located where the first and second sorbentmaterials overlie one another. The first-ligand binding molecules at thetest site may be arranged in one or more lines or other distinctivepatterns. A control line or site may be provided downstream from thetest site.

In one embodiment, the second-ligand binding molecules are secondconjugates that include immobilized ligand binding molecules conjugatedwith particles. In one embodiment, the second conjugate include antigensconjugated with particles. In one embodiment, the particles conjugatedwith the antigens comprise white latex. In another embodiment, thesecond conguate includes antibodies conjugated with particles. In oneembodiment, the particles conjugate with the antibodies comprise whitelatex. In one embodiment directed to detecting influenza (“flu”), thesecond-ligand binding molecules include antigens of at least oneinfluenza (“flu”) antigen and the test site is provided with immobilizedantigen of at least one influenza antigen different but related to theat least one flu antigen of the immobilized conjugate. In oneembodiment, the first conjugate is a gold sol conjugated to protein A.

In another embodiment a dual path immunoassay test cell device fordetecting the presence of a first ligand in a sample is provided with afirst sorbent material defining a first horizontal flow path and asecond sorbent material distinct from the first sorbent material anddefining a second horizontal flow path, the first and second sorbentmaterials overlying one another at a test site. The first flow path hasa first location for receiving a first solution, which, in the case of aliquid conjugate system is a conjugate solution, and which, in the caseof a dry conjugate system is a buffer solution. Where a buffer solutionis utilized, the first sorbent material is provided with a first(mobile) conjugate located downstream of the first location. The secondflow path has a second location for receiving a second solutioncomprising a sample such as blood, urine, saliva, or other sample thathas been previously mixed with second-ligand binding molecules and, ifdesired, buffer and optionally filtered prior to being applied as thesecond solution to the second location. Where the sample has been mixedwith second-ligand binding molecules and not filtered, in oneembodiment, the second flow path may include a filter for the secondsolution. The second-ligand binding molecules are related to the firstligand for which the sample is being tested but are not the same and inone embodiment may include immobilized ligand binding molecules such asantigens or antibodies conjugated with particles such as latex. In oneembodiment directed to detecting influenza (“flu”), the second ligandbinding molecules include antigens of at least one influenza (“flu”)antigen and the test site is provided with immobilized antigen of atleast one influenza antigen different but related to the at least oneflu antigen of the immobilized conjugate. In one embodiment the testsite is provided with first-ligand binding molecules such as immobilizedantigens or antibodies or other molecules such as aptamers, nucleicacids, etc. located where the first and second sorbent materials overlieone another. The first-ligand binding molecules at the test site may bearranged in one or more lines or other distinctive patterns. A controlline or site may be provided downstream from the test site.

In one aspect, the second-ligand binding molecules are used as adepleting mechanism that captures and thereby depletes antibodies (orantigens) related to the antibodies (or antigens) that are beingdetected at the test site. By way of example, where the test siteincludes a pendemic flu-A antigen for identifying the presence of aflu-A antibody in the sample, the second conjugate may be provided withone or more common flu-A antigens and or flu-B antigens. In this manner,common flu-A and flu-B antibodies in the sample that may otherwise becaptured or retained at the test site (because of their structure whichcan be similar in many ways to the related pandemic flu-A antibodies)are generally captured by the second immobilized conjugate; i.e., thenumber of common flu-A and flu-B antibodies reaching the test site isdepleted. As a result, the sensitivity of the test is increased.

In one aspect, the use of a white latex conjugate as the immobilizeddepleting conjugate reduces the visibility of the conjugate should it beloosened and travel with the sample to the test site and arrive at thetest site.

Where the test cell is provided in a housing, the housing is providedwith a first opening adjacent the first location and a second openingadjacent the second location. A viewing window is provided in thehousing above the test line. Similarly, a viewing window may be providedin the housing above the control line.

According to one set of embodiments, the sorbent materials are laid outin a T shape, where the first location for receiving the buffer orbuffer-conjugate solution is located near one end of the top bar of theT, the second location for receiving the sample is located near the endof the stem of the T, and the sorbent materials overlie each other atthe intersection. Of course, the sorbent materials may be laid out inother configurations, and the housing may take other shapes, such asrectangular, square, irregular, etc. regardless of the manner in whichthe sorbent materials are arranged.

In one embodiment of the invention, the materials, thicknesses andlengths of the first and second sorbent materials are chosen to adjustthe timing regarding the liquid sample and liquid buffer reaching thetest site.

In the dry conjugate system, a first dry conjugate is provided betweenthe first opening and the test site. The first conjugate is supported onor within the sorbent material such that when a buffer is added in thefirst opening, the sorbent material wicks the buffer to the firstconjugate which is then carried by the buffer to the test site. In theliquid conjugate system, a buffer-conjugate liquid subsystem is providedand applied to the first opening. The sorbent material then wicks thebuffer-conjugate subsystem to the test site.

In another embodiment a dual path immunoassay test cell device fordetecting the presence of a first ligand in a sample is provided with afirst sorbent material defining a first horizontal flow path and asecond sorbent material distinct from the first sorbent material anddefining a second horizontal flow path, the first and second sorbentmaterials overlying one another at a test site. The first flow path hasa first location for receiving a first solution, which, in the case of aliquid conjugate system is a conjugate solution, and which, in the caseof a dry conjugate system is a buffer solution. Where a buffer solutionis utilized, the first sorbent material is provided with a first(mobile) conjugate located downstream of the first location. The firstconjugate includes a marker such as a colored latex or particle and afirst interim binding agent such as (by way of example only)streptavidin or an anti-biotin antibody. The second flow path has asecond location for receiving a second solution comprising a sample suchas blood, urine, saliva, or other sample that has been optionallypreviously mixed with second-ligand binding molecules and, if desired,buffer and is optionally filtered to remove the second-ligand bindingmolecules and second ligand bound thereto prior to being applied as thesecond solution to the second location. The second flow path is providedwith immobilized first-ligand binding molecules. The immobilizedfirst-ligand binding molecules may include a second conjugate of latexparticles (e.g., white latex) to which are bound antibodies or antigensand a second interim binding agent such as biotin. In this manner, whenthe sample includes the first ligand, the first-ligand binding moleculeswith the first ligand and second interim binding agent attached theretoare carried by the filtered sample solution to the test site along thesecond flow path. The test site which is located where the first andsecond sorbent materials overlie one another is provided with animmobilized binding agent which bind to the antigen or antibodies of thesample. Thus, the ligand with the second interim binding agent is boundat the test site, and when the first conjugate travels down the firstflow path with the colored latex or particle and first interim bindingagent, the interim binding agents will attach and keep the colored latexat the test site. A control line or site may be provided downstream fromthe test site.

In one aspect, where the first flow path is provided with a conjugatehaving a the second flow path is provided with immobilized first-ligandbinding molecules with a second interim binding agent and the first testline is provided with a conjugate having a first interim binding agent,and sensitivity of the test is enhanced.

According to one method, a system for detecting the presence of a firstligand in a sample is provided and includes a test cell having a firstsorbent material having a first location for receiving a buffer solution(in the case of a dry conjugate system) or a conjugate solution (in thecase of a liquid conjugate system) with the first sorbent materialdefining a first horizontal flow path, and a second sorbent materialhaving a second location for receiving a sample and defining a secondhorizontal flow path distinct from the first flow path, with the secondsorbent material having a second-ligand binding molecules locateddownstream of the second location, and a test line or test site withimmobilized first-ligand binding molecules such as antigens, antibodies,aptamers, nucleic acids, etc. located in a test zone at a junction ofthe first and second sorbent materials. If desired, a housing is alsoprovided having a first opening for receiving the buffer or conjugatesolution, a second opening for receiving the sample, and a viewingwindow above the test line. A sample of interest is provided to thesecond opening or location and permitted to migrate down to the testline over time. After a desired amount of time, a liquid such as abuffer solution is added to the first opening or location. If the firstsorbent material is supporting a conjugate (i.e., in a dry conjugatesystem), the liquid can be simply a buffer solution. If the firstsorbent material is not supporting a conjugate (i.e., in a liquidconjugate system), the liquid can be a buffer-conjugate liquidsubsystem. In any event, after sufficient time to permit the firstconjugate to migrate to the test site (and control site if provided),the test site (and control site if provided) is inspected in order todetermine whether the sample is “positive” or not.

According to another method, a system for detecting the presence of afirst ligand in a sample is provided and includes a test cell having afirst sorbent material having a first location for receiving a buffersolution (in the case of a dry conjugate system) or a conjugate solution(in the case of a liquid conjugate system) with the first sorbentmaterial defining a first horizontal flow path, and a second sorbentmaterial having a second location for receiving a sample and defining asecond horizontal flow path distinct from the first flow path with anoptional filter, and a test line or test site with immobilizedfirst-ligand binding molecules such as antigens, antibodies, aptamers,nucleic acids, etc. located in a test zone at a junction of the firstand second sorbent materials. If desired, a housing is also providedhaving a first opening for receiving the buffer or conjugate solution, asecond opening for receiving the sample, and a viewing window above thetest line. A sample of interest is provided to a mixing chamber havingsecond-ligand binding molecules and optional buffer. The sample is mixedwith the second-ligand binding molecules (and buffer) and optionallyfiltered to remove the second-ligand binding molecules and second ligandattached thereto if the second flow path has no filter. The optionallyfiltered sample is provided to the second opening or location andpermitted to migrate along the second flow path down to the test site.After a desired amount of time, a liquid such as a buffer solution isadded to the first opening or location. If the first sorbent material issupporting a conjugate (i.e., in a dry conjugate system), the liquid canbe simply a buffer solution. If the first sorbent material is notsupporting a conjugate (i.e., in a liquid conjugate system), the liquidcan be a buffer-conjugate liquid subsystem. In any event, aftersufficient time to permit the first conjugate to migrate to the testsite (and control site if provided), the test site (and control site ifprovided) is inspected in order to determine whether the sample is“positive” or not.

According to another method, a system for detecting the presence of afirst ligand in a sample is provided and includes a test cell having afirst sorbent material having a first location for receiving a buffersolution (in the case of a dry conjugate system) or a conjugate solution(in the case of a liquid conjugate system) with the first sorbentmaterial defining a first horizontal flow path for a first conjugatehaving a marker and a first interim binding agent, and a second sorbentmaterial having a second location for receiving a sample and defining asecond horizontal flow path distinct from the first flow path withimmobilized first-ligand binding molecules such as antibody or antigenbound to a second interim binding agent, and a test line or test sitewith immobilized binding agent located in a test zone at a junction ofthe first and second sorbent materials. If desired, a housing is alsoprovided having a first opening for receiving the buffer or conjugatesolution, a second opening for receiving the sample, and a viewingwindow above the test line. A sample of interest is optionally providedto a mixing chamber having second-ligand binding molecules and optionalbuffer. The sample may be mixed with the second-ligand binding molecules(and buffer) and filtered to remove the second-ligand binding moleculesand second ligand attached thereto. The optionally filtered sample isprovided to the second opening or location and may then interact with asecond conjugate having a second interim binding agent as it migratesalong the second flow path to the test site. After a desired amount oftime, a liquid such as a buffer solution is added to the first openingor location. If the first sorbent material is supporting a firstconjugate (i.e., in a dry conjugate system), the liquid can be simply abuffer solution. If the first sorbent material is not supporting aconjugate (i.e., in a liquid conjugate system), the liquid can be abuffer-conjugate liquid subsystem containing the first conjugate. In anyevent, after sufficient time to permit the second conjugate to migrateto the test site (and control site if provided), the test site (andcontrol site if provided) is inspected in order to determine whether thesample is “positive” or not.

It will be appreciated that the systems can be used in conjunction withdifferent types of samples such as blood, urine, saliva, etc. The samplemay be diluted or mixed with buffer prior to being added through thesecond hole. Alternatively, in some cases, the sample may be addedthrough the hole and then a diluent may be added through the same hole.

Objects and advantages will become apparent to those skilled in the artupon reference to the detailed description taken in conjunction with theprovided figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a top schematic view of a first embodiment.

FIG. 1A is a cross-sectional view taken along line 1A-1A of FIG. 1.

FIG. 1B is a cross-sectional view taken along line 1B-1B of FIG. 1.

FIG. 2A is a chart comparing test results of the apparatus of FIG. 1against the test results of a standard dual path platform apparatus andshowing the depletion of non-pandemic flu antibodies by the apparatus ofFIG. 1

FIG. 2B is a chart comparing test results of the apparatus of FIG. 1against the test results of a standard dual path platform apparatus andshowing non-depletion of flu-B antibodies by the apparatus of FIG. 1.

FIG. 3 is a diagram showing a kit including a water vial, a vial withconjugate, a vial with diluent, a blood collection and transfer device,three transfer pipettes, and a filter chamber.

FIG. 4A is a diagram depicting a first alternative embodiment.

FIG. 4B is a diagram depicting a second alternative embodiment.

FIG. 4C is a diagram depicting a third alternative embodiment.

DETAILED DESCRIPTION

Turning now to FIGS. 1, 1A and 1B, an immunoassay device test cell 10for testing for the presence of a first ligand in a sample is providedand includes a housing 20 having a top wall 21 defining first and secondholes 24, 26, and a window 28, and first and second sorbent or bibulousmaterials 30, 32 defining perpendicular horizontal or lateral flow pathsin the housing. The first sorbent material 30 includes a plurality ofzones and may be made from a plurality of materials. A first zone 31(sometimes called a filter zone) is located at the first hole 24 andextends to a second zone 33 (sometimes called a test zone) which islocated at the junction of a “T”. The first zone 31 preferably includesa filter 31 a, a pad 31 b on or in which a conjugate 39 having desiredantigens or antibodies with attached colored markers is deposited andimmobilized, and a first portion of a thin membrane or sorbent orbibulous material 30 typically made from nitrocellulose with a plasticbacking (not shown). In one embodiment, and by way of example only,conjugate 39 may be a gold sol conjugated to protein A. The first zone31 is adapted to receive a buffer solution, to cause the buffer solutionto contact the conjugate, thereby mobilizing the conjugate, and to wickthe conjugate-carrying buffer solution to the second zone 33. The second(test) zone 33 includes a second portion of the thin membrane 30 whichcan be printed with a test line 50 having immobilized first ligandbinding molecules such as antigens or antibodies (depending on whetherthe test cell is designed to test for the presence of antibodies orantigens) on the membrane as is well known in the art. The test line 50may be seen through the window 28 of clear plastic provided in thehousing. A third zone 35 (sometimes called a control zone) whichincludes a third portion of the thin membrane 30 may also be printedwith a control line 60 typically containing antibodies to the conjugateantigens (or in some cases antibodies which will bind to conjugateantibodies, or even antigens which will bind to conjugate antibodies) asis well known in the art. Where the third zone 35 is provided, thewindow 28 extends above the control line 60. If desired, a fourth zone37 (sometimes called a reservoir zone) may be provided as a wickingreservoir as is also well known in the art. The fourth zone 37 includesa relatively thicker absorbent paper. Preferably overlying all the zonesis a thin, preferably transparent plastic film or card 38 a having anadhesive which keeps the sorbent materials in place. The card 38 a maybe cut with an opening at hole 24 so that it does not block liquidaccess to the hole 24.

The second sorbent material 32 may also be made from a plurality ofmaterials and include a plurality of zones. The first zone 62 (sometimescalled a filter zone) includes a filter or pad 32 a and a pad 32 b on orin which second-ligand binding molecules are provided and immobilized,where the second ligand is different than but related to the firstligand, and a first portion of a thin membrane or sorbent or bibulousmaterial 32 typically made from nitrocellulose with a backing (notshown). The second-ligand binding molecules may include antigens orantibodies or other molecules such as aptamers, nucleic acids, etc. thatbind to ligands that are similar to but different than the firstligands. The second-ligand binding molecules may be provided as aconjugate 41 having desired antigens or antibodies with attachedparticles. The first zone 62 is located at the second hole 26 andextends to the second zone 63. The second zone 63 includes a secondportion of the thin membrane 32 which is in contact with the second zone33 of the first sorbent material 30. As is seen in FIGS. 1A and 1B, thefirst sorbent material 30 overlies the second sorbent material 32 suchthat the membranes are in contact with each other (as opposed to thebackings contacting the membranes or each other), and such that the testline 50 is effectively located between the membranes. Thus, test line 50could be printed on the second zone 63 of the second sorbent material 32instead of, or in addition to the second zone 33 of the first sorbentmaterial 30. If desired, a thin plastic film or card 38 b having anadhesive which keeps the second sorbent material in place may beutilized. With the provided arrangement it takes time for the sample totravel from its application point to the second zone 63 and the testsite, and application of sample to the second flow path does notimmediately wet the test site.

In one embodiment the conjugate 41 on the conjugate pad 32 b includesantigens conjugated with a particle that is not readily visible to thehuman eye against the background of the test area. In one embodiment,the particle is a white latex. One embodiment of a white latex is a 0.32micron white latex bead available from Thermo Fisher Scientific, Inc.,Holtsville, N.Y. The antigens of conjugate 41 are different than but arerelated to the antigens of test line 50. By way of example only, in anembodiment directed to detecting pandemic influenza (“flu”), the secondconjugate includes antigens of at least one influenza (“flu”) antigen(e.g., two different flu A antigens such as H1 and H3 flu antigens) andthe test site is provided with immobilized antigen of at least thepandemic influenza antigen of interest which is different from butrelated to the at least one flu antigen of the immobilized conjugate 41.In another embodiment, the second conjugate includes antibodiesconjugated with white latex and the test site 50 includes antibodiesdifferent than but related to the antibodies of the conjugate 41.

In one aspect, the second conjugate is used as a depleting mechanismthat captures and thereby depletes antibodies related to the antibodiesthat are being detected at the test site. By way of example, where thetest site includes a flu-B antigen for identifying the presence of aflu-B antibody in the sample, the second conjugate may be provided withone or more flu-A antigens; i.e., there may be a plurality of slightlydifferent second conjugates. In this manner, flu-A antibodies in thesample that may otherwise be captured or retained at the test site(because of their structure which can be similar in many ways to therelated flu-B antibodies) are generally captured by the secondimmobilized conjugate; i.e., the number of flu-A antibodies reaching thetest site is depleted. As a result, the sensitivity of the test isincreased. It will be appreciated that the test site could include aflu-A antigen for identifying the presence of a particular flu-Aantibody in the sample, and the second conjugate may be provided withone or more flu-B antigens and one or more flu-A antigens that aredifferent from but related to the particular flu-A antigen at the testsite. Further, it will be appreciated that the test site may be providedwith more than one test line, containing different flu antigens. Thoseflu antigens could include a plurality of flu-A antigens, a plurality offlu-B antigens, or one or more flu-A and one or more flu-B antigens. Thesecond immobilized conjugate will be adjusted accordingly to includeconjugate that will deplete those antigens that are related to theantigens of the test lines but are not the subject of the test.

In one aspect, the use of a white latex conjugate as the immobilizeddepleting conjugate reduces the visibility of the conjugate should it beloosened and travel with the sample to the test site and get captured atthe test site. In another aspect, latex beads of a size larger than thepore size of the second migration path may be utilized in order toprevent movement of the conjugate along the second migration path.

Where standard-type nitrocellulose strips with a backing are utilized asthe first and second membranes, the membranes can have different poresizes. For example, if membrane 31 (for the first conjugate migration)has a 3 μ pore size, and membrane 32 (for the sample migration) has a 15μ pore size, sample applied to membrane 32 will tend to migrate and stayin the sample membrane 32 and will tend not to migrate into theconjugate membrane 31.

The immunoassay of FIGS. 1, 1A and 1B is preferably utilized as follows.First, a sample (not shown) possibly containing antibodies (or antigens)is optionally diluted (e.g., with buffer) and provided to the secondopening or hole 26. The sample does not immediately wet the test sitebut is allowed to take time to migrate from pad 32 a to conjugate pad 32b and then from zone 61 of the second sorbent material 32 to its secondzone 63 which is contact with the second zone 33 of the first sorbentmaterial 30. If the sample is not first diluted, optionally, afterproviding the sample to hole 26, a measured amount of liquid such as abuffer solution may be added to hole 26 to help in the migration of thesample. Regardless, if the sample includes antigens or antibodies thatreact with the second conjugate 41 of conjugate pad 32 b, those antigensor antibodies are captured by the conjugate 41 and are depleted from thesample before reaching the test line 50 which is printed atop the secondzone 33 of the first sorbent material or infused therein. To the extentthat the conjugate 41 loosens from the pad 32 b and travels alongmembrane 32 down to the test site and is captured there, the conjugate41 will not be particularly visible because the white latex particleswill not be seen on the white background of the test site. Regardless,after a desired amount of time, by which time the antibodies (orantigens) in the sample (if present) will have had an opportunity tobind to the antigens (or antibodies) immobilized at the test line 50, aliquid such as a buffer solution (not shown) is added to the firstopening 24. After another period of time, sufficient to permit thebuffer solution to cause the conjugate to migrate to the test site 50(and control site 60 if provided), and to bind with the antigens (orantibodies) of the sample that are captured at the test site 50 (ifany), the test site (and control site 60 if provided) is inspected viawindow 28 in order to determine whether the sample is “positive” or not.Typically, a “positive” test indicating the presence of the antibody (orantigen) in the sample is obtained when both the test site 50 and thecontrol site 60 show lines of color. A “negative” test indicating thelack of the presence of the antibody (or antigen) in the sample isobtained when only the control site 60 shows a line of color.

The use of the apparatus may be expedited by providing the housing withnumbering and/or lettering to indicate that hole 26 is for receiving thesample (and optionally some buffer) and is to be used first, and thathole 24 is for receiving the buffer solution and is to be used second.

Those skilled in the art will appreciate that the immunoassay 10functions as follows. Because the test line 50 is provided with antigens(or antibodies) immobilized on a membrane, if the test sample containsantibodies to the antigens (or antigens to the antibodies), theantibodies (or antigens) will bind themselves to the antigens (orantibodies) at the test line. Because the test sample passes through aconjugate pad 32 b having immobilized second conjugate 41 with antigens(or antibodies) that are related to but different than the antigens (orantibodies) of the test line, related antibodies or antigens to thosebeing tested, if present, will be captured by the congugate 41 and heldat the conjugate pad 32 b, and when the test sample reaches the testline, the antibodies (or antigens) of the sample, if present, will bindto the antigen (or antibody) at the test line. Because the relatedantibodies (or antigens) are depleted, they will not reach the testline, and if they do, they will already be conjugated with a latex thatwill reduce their activity at the test site. Regardless, the test sitewill be more specific to the antibodies or antigens whose presence is tobe detected. After the sample has reached the test site, the firstconjugate 39 containing an antigen for the antibody (or antibody for theantigen) coupled to a colored marker is caused to migrate to the testline. If the test sample contains the antibodies (or antigens) which arenow held at the test line 50, the antigen (or antibody) of the conjugatewill bind itself to the antibodies (or antigens) and the colored markerwill cause a colored line to appear at the test site 50. If the testsample does not contain antibodies (or antigens), the conjugate will nothave the antibodies (antigens) to bind to at the test line 50, and nocolored line will appear at the test site 50. On the other hand, becausethe control line 60 is provided with antibodies (or antigens), theantigens (or antibodies) of the conjugate will always bind to theantibodies (or antigens) in the control line 60, thereby causing acolored line to appear at the control site 60 if the conjugate reachesthe control site 60. Thus, if sufficient buffer solution is provided tothe test cell, a colored line should always appear at the control site60, thereby providing a control for the test.

Turning to FIG. 2A, it can be seen that the apparatus of FIGS. 1, 1A and1B can provide improved test results relative to a standard dual pathplatform apparatus such as described and shown in previouslyincorporated U.S. Pat. No. 7,189,522. In particular, three sets of fivetest apparatus such as described above with reference to FIGS. 1, 1A and1B were prepared with a second conjugate pad 32 b provided with aconjugate 41 having H3 and H1 flu-A antigen conjugated with beads, and aDPP test line provided with Flu A antigens. One set of five apparatusutilized magnetic beads separately conjugated with H1 antigen and H3antigen (H1+H3 Mag). A second set utilized latex beads separatelyconjugated with H1 and H3 antigen (H1+H3 Latex). A third set utilizedlatex beads with combined H1 and H3 conjugation (H1/H3 Latex).Similarly, a set of devices such as described and shown in previouslyincorporated U.S. Pat. No. 7,189,522 were provided (No Ad) with a testline having the same flu-A antigens. Test samples from five differentindividuals having H3 antibodies were prepared and applied to the secondflow paths of the sets of devices described above with reference toFIGS. 1, 1A and 1B and the set of devices of U.S. Pat. No. 7,189,522.After waiting for the samples to reach the test sites, buffer was addedto the first migration path of each device to move the marker conjugateto the test sites. The intensity of the signals at each test site wasmeasured and plotted. As seen in FIG. 2A, the test lines of the fivestandard dual path platform apparatus (No Ad) showed a relativeintensity (with a digital reader) ranging from about 700 to well over4000 compared to a relative intensity of nearly zero for the apparatusof FIGS. 1, 1A, and 1B utilizing the beads for the magnetic and latexbeads. These test show that the apparatus of FIG. 1 is successful indepleting the flu-A antibodies by utilizing the flu-A antigen—particleconjugate in the flow path of the sample. Where white particles areutilized, to the extent that any flu-A antigen—particle conjugate wascarried down to the test site and captured there, the white particleprevents the conjugate from being seen against the white background ofcard 38 b over which the test line 50 is located. It should beappreciated that by depleting flu-A H1 and H3 (seasonal flu) with thelatex conjugate system in the path of the sample, the sensitivity andspecificity of the test with a test line for pandemic flu A will beincreased because of the elimination of the cross-reactivity between theseasonal and pandemic flu A antigens.

In one embodiment, the conjugate in the sample flow path utilizesfragments or fractions of seasonal flu H1 and H3 conjugated to latexparticles. The fragments are immunodominant portions of the particlethat will not substantially cross-react with other flu antigens and aredifferent from the H1 and H3 antibodies that might be used as captureantibodies at the test site in the membrane (the whole molecule of H1and H3). As a result, when a test for pandemic flu is provided with atest line including pandemic flu antibodies, the H1 and H3 fragmentconjugates will have minimal cross-reactivity with pandemic flu antigensresulting in a better detection of a pandemic flu at the test line.

Turning to FIG. 2b , other samples were prepared having flu-B/Brisantibodies. The samples were applied to a sets of the standard dual pathplatform apparatus such as described in previously incorporated U.S.Pat. No. 7,189,522 where the test line had flu-B/Bris antigen (No Ad)and to sets of devices such as shown in FIGS. 1, 1A and 1B where thesecond conjugate pad 32 b was provided with a conjugate 41 having H1 andH3 flu-A antigens conjugated to beads, and a test line provided withflu-B/Bris antigens. As with tests of FIG. 2A, one set of apparatusutilized magnetic beads separately conjugated to H1 and H3 (H1 +H3 Mag),a second set utilized 0.32 micron white latex beads separatelyconjugated (H1+H3 Latex), while a third set utilized the white latexbeads with combined conjungation (H1/H3 Latex). As seen in FIG. 2B, thepositive results at the test line of the apparatus 10 of FIG. 1 is justas strong as the test lines of the standard dual path platform apparatusshowing that the conjugate 41 located in the second migration path didnot interfere with the results, as the signals at the test lines werenearly the same for all tests of a particular sample. Taking FIGS. 2Aand 2B together, it will be appreciated that the apparatus 10 of FIGS.1, 1A, and 1B has higher sensitivity.

Turning now to FIG. 3, a kit 100 is seen that includes a water vial 101with water 102, a vial 103 with freeze dried latex conjugate 104, adiluent vial 105 with a diluent 106, a blood collection and transferdevice 107, four transfer pipettes 108 a, 108 b, 108 c, 108 d, and afilter chamber assembly 109. It will be appreciated that the kit couldhave different numbers of elements. Thus, rather than separatelymaintaining water and freeze dried latex conjugate, a “wet” latexconjugate may be stored utilizing water and/or diluent. Likewise, ratherthan maintaining a vial of diluent, diluent may be provided as part ofthe “wet” latex conjugate. Also, rather than utilizing four transferpipettes, fewer transfer elements may be utilized. In one embodiment,kit 100 may be used in conjunction with an immunoassay device test cellsuch as device 10 of FIGS. 1, 1A, and 1B. In another embodiment, kit 100may be used in conjunction with other immnoassay devices such as ELISA(enzyme-linked immunosorbent assay). In another embodiment, kit 100 maybe used in conjunction with an immunoassay device test cell such asdescribed in previously incorporated U.S. Pat. No. 7,189,522.

More particularly, the water 102 in vial 101 may be mixed with thefreeze dried latex conjugate 104 in vial 103 by using a pipette 108 aand transferring the water to the latex vial. The vial 103 may beinverted multiple times in order to cause the freeze dried latexconjugate to be reconstituted. The reconstituted latex may be stored ina refrigerator if desired. In one embodiment, the dried latex conjugateis a conjugate of one or more flu antigens such as H1 and H3 withmicrobeads of latex. The latex beads may be of an easily visible color,e.g., blue.

When it is desired to test a sample, the sample, e.g., blood, may beobtained from a patient in a desired manner, e.g., a fingerstick,utilizing a blood collection and transfer device 107 such as a MinivettePOCT manufactured by Sarstedt, Newton, North Carolina. The blood samplemay be transferred into the diluent vial 105 containing a diluent 106such as heparin or EDTA. The reconstituted latex conjugate may then betransferred into the diluent vial 105 by using a pipette 108 b, and theblood and reconstituted latex conjugate may be mixed by invertingmultiple times over a period of time and also giving antibodies in theblood an opportunity to be captured by the latex conjugate. Aftersufficient mixing and a sufficient period of time, the contents of thesample diluent vial 105 may then be transferred with pipette 108 c to afilter chamber 109 such as a GE Healthcare Life Sciences Mini-UniPrepfilter chamber comprising a filter 109 a, compressor 109 b, plunger 109c, and a tube 109 d, although other filter mechanisms could be utilized.Using the hand compressor 109 b of the filter chamber, the filter 109 acan be plunged into the sample mixture, and the filtered sample can becollected in the tube 109 d of the filter chamber. It will beappreciated that the filter is chosen to have pores that are smallerthan the size of the latex conjugate beads. As a result, the conjugatebeads (with captured antibodies, if any) are filtered out of the sample,and the sample (with antibodies that haven't been captured by theconjugate) with the previously added diluent and water will be caught inthe tube 109 d. Thus, while the contents of the sample diluent vial 105that were transferred to the filter chamber 109 may have appeared to bedark blue (due to the blue latex conjugate and the blood), the contentsof the tube 109 d should be light red (the color of diluted blood).Regardless, it will be appreciated that the ligands that are related tobut not the same as the ligands of interest will have been removed fromthe sample.

The contents of tube 109 d are then transferred by pipette 108 d andused in conjunction with an immunoassay device. In one embodiment, theimmunoassay device is an otherwise prior art type device such as ELISA(enzyme-linked immunosorbent assay) or a LUMINEX assay sold by ThermoFisher Scientific, Holtsville, N.Y. When provided with a sample that isprocessed in this manner, the results of the ELISA and the LUMINEXdevices are enhanced. In another embodiment, the immunoassay device towhich the contents of tube 109 d are transferred is an immunoassaydevice test cell such as described in previously incorporated U.S. Pat.No. 7,189,522 such as by applying a selected amount of the contents tothe (second) location for receiving the liquid sample, waiting for theliquid sample to reach the test site via the second migration path, andthen applying buffer or a buffer—conjugate subsystem to the firstlocation to cause a conjugate to reach the test site via the firstmigration path. When provided with a sample that is processed aspreviously described, the results of the device described in previouslyincorporated U.S. Pat. No. 7,189,522 are enhanced.

In another embodiment, rather than utilizing a kit 100 with elementssuch as a water vial, a vial with freeze dried latex conjugate, adiluent vial, a filter chamber assembly, etc., the kit includes aconjugate which may be maintained in a wet form with or without buffer,or may be maintained in a freeze-dried conjugate format which may bereconstituted with water and/or a buffer solution. In one embodiment,the latex conjugate comprises white latex beads with antibodies orantigens conjugated thereto. The sample and conjugate are mixed togetherto permit the conjugate to deplete interfering antigens or antibodies.The mixed sample and conjugate may then be applied to an immunoassaydevice test cell such as described in previously incorporated U.S. Pat.No. 7,189,522 such as by applying a selected amount of the contents tothe (second) location for receiving the liquid sample, waiting for themixed sample and conjugate to reach the test site via the secondmigration path, and then applying buffer or a buffer—conjugate subsystemto the first location to cause a conjugate to reach the test site viathe first migration path. When provided with a sample that is processedas previously described, the results of the device described inpreviously incorporated U.S. Pat. No. 7,189,522 are enhanced.

Turning to FIGS. 4A-4C, additional embodiments are provided that resultin an apparatus having an enhanced test signal. FIGS. 4A-4C aredescribed with reference to HIV test devices although they are notlimited thereto. The embodiments of FIGS. 4A and 4B are similar to thatof FIGS. 1, 1A, and 1B except that the conjugates provided on pads 31 band 32 b are different, and the immobilized test line antigen is an HIVantibody rather than a flu antibody. More particularly, in FIG. 4A,conjugate 41 a in the sample migration path 32 includes a latex particle(e.g., a white latex) to which a MAb-1 p24 antibody and a first interimbinding agent (e.g., biotin antigen) are conjugated. The test line 50 isprovided with a monoclonal anti-HIV antibody protein (MAb-2 p24). Thebuffer-conjugate subsystem of the first migration path 30 is providedwith a conjugate 39 a including a marker (e.g., blue latex or gold sol)and a second interim binding agent (e.g., streptavidin) conjugatedthereto that is chosen to bind to the first interim binding agent. Withthe provided system, when a sample containing HIV p24 antigen is addedto the test apparatus through hole 26, the HIV p24 antigen in the samplewill bind to the MAb-1 p24 of the conjugate, and the sample with theantigen of interest bound to the conjugate will travel to the test line50 where the p24 antigen of the sample will be caught by the MAb-2 p24antibody at the test line. When buffer is added to the first sorbentstrip through hole 24, the marker conjugate will move to the test linewhere the first interim binding agent will bind with the second interimbinding agent, and the marker will appear at the test line.

The embodiment of FIG. 4B is very similar to the embodiment of FIGS. 4A,except that instead of the second interim binding agent of conjugate 39a being a tetrameric protein such as streptavidin, the second interimbinding agent is an anti-biotin antibody. As a result, where the samplecontains HIV p24 antigen, at the test line, the HIV p24 antigen will beretained at the test line by the MAb-2 p24 antibody of the test line,and the marker conjugate will bind to the first conjugate because theantibiotin antibody will bind to the biotin that is part of the firstconjugate as seen in FIG. 4B

The embodiment of FIG. 4C is likewise similar to the embodiments ofFIGS. 4A and 4B, except that a double interim binding arrangement isutilized. More particularly, the second sorbent material 32 is providedwith a pad 32 c in addition to pad 32 b. In one embodiment, pad 32 b isprovided with MAb-1 HIV p24 antigen conjugated with biotin 41 x with thebiotin acting as a first interim binding agent of a first pair, and pad32 c is provided with particles such as a white latex particlesconjugated with streptavidin and a secondary antigen such as FITC-A2(fluorescein isothiocyanate) 41 y. The streptavidin of particles 41 yact as a second interim binding agent of a first pair, and the FITC-A2acts as a first interim binding agent of a second pair. Pad 31 b isprovided with a conjugate 39 z having a marker to which is conjugated ananti-FITC antibody which acts as a second interim binding agent of asecond pair. With the provided arrangement, if the sample contains a p24antigen, when the sample is added to the second sorbent material 32, thep24 antigen will attach to the MAB-1 HIV p24 antibody with biotin at pad32 b. As the sample progresses along its migration path to pad 32 c, thebiotin will bind to the streptavidin of the conjugate 41 y; i.e., thefirst and second interim binding agents of the first pair bind together,and the complex of the p24 antigen—MAB-1 HIV p24 antibody withbiotin—streptavidin/white latex/FITC antigen conjugate 41 y will move tothe test site that includes MAB-2 HIV p24 antibody. At the test site,the p24 antigen of the sample will bind to the MAB-2 HIV p24 antibody ofthe test site, and the entire previously—described complex will be heldat the test site. When buffer is then added to the first migration pathand marker-anti-FITC antibody conjugate is moved to the test site, theanti-FITC antibody will bind to the FITC-A2 being held at the test site;i.e., the first and second interim binding agents of the second pairbind together. As a result, the marker will be held at the test line andprovide a positive test result.

The embodiments of FIGS. 4A-4C may all be used in conjunction with asample being provided directly to the apparatus or with a sample such asthe previously described sample contained in tube 109 d which hasresulted from a sample having been previously mixed with a depletionconjugate for antigens or antibodies different from but related to theantigen or antibody of interest and then filtered. In all cases, themolecules and conjugates on pads 32 b and 31 b, and 32 c (if present)are appropriately selected, as are the molecules on the test line 50 andthe freeze-dried depletion conjugate 104.

There have been described and illustrated herein several embodiments ofimmunoassays and methods of their use. While particular embodiments havebeen described, it is not intended that the claims be limited thereto,as it is intended that the claims be as broad in scope as the art willallow and that the specification be read likewise. Thus, while thespecification discusses ligand binding using antigen/antibody reactions,other ligand binding mechanisms such as aptamer binding, nucleic acidbinding, enzymatic binding, etc. may also be used. Also, while the testcells are described as having a single line for testing for a singleligand, it will be appreciated that two or more lines may be utilizedfor testing for more than one ligand. Further, while the test cells aredescribed as having holes in the top wall of a housing for receiving thesample and the buffer-solution or buffer-conjugate subsystem, it will beappreciated that one or both holes may be provided in the end wall orside wall of the housing. Similarly, while the sorbent material wasdescribed as preferably including a thin plastic backing, it will beappreciated that the plastic backing could be provided only at certainlocations or not be provided at all. Where only partial backings or nobackings are provided, the test and control sites can be located oneither or both sides of the sorbent material. Further yet, while a teststrip and control strip are shown is being rectangular in configuration(i.e., lines), it will be appreciated that the test and control sitescan be configured differently such as in circles, squares, ovals, abroken line, etc. In fact, the test site and control site can beconfigured differently from each other.

Those skilled in the art will also appreciate that the housing may bemodified in additional ways to include separate windows for each testline. Also, while the embodiments were described in conjunction with theuse of a buffer solution which is added to the migration path of theconjugate and optionally to the migration path of the sample, it will beappreciated that that one or more buffers may be chosen as desired to beadded to the migration paths depending upon the test or tests to beconducted. Thus, buffers such as phosphate buffers or TRIS (trishydroxymethylaminomethane) buffers are often utilized. However, theembodiments are intended to encompass the use of any diluent includingwater. In addition, the diluent may, if needed, may be added to andmixed with the sample prior to adding the sample to the sorbent materialor the sample may be deposited first and the diluent may be addedthereafter. Likewise, any diluent capable of causing the conjugate ofthe “non-sample” path to migrate may be utilized, and may be premixedwith the conjugate in a liquid conjugate system, or provided to themigration path for the conjugate in a dry conjugate system.

Those skilled in the art will also appreciate that while the embodimentswere described with particular reference to detection of a flu antibodyand HIV p-24 antigen, the apparatus and methods may be useful indetection of other antibodies or antigens whether human or animal. Also,while the embodiments were described with particular reference to theuse of blood as a sample, it will be appreciated that other body fluidsor excretions, or blood portions may be utilized including, but notlimited to urine, feces, saliva, spitum, blood serum (plasma), etc. Itwill therefore be appreciated by those skilled in the art that yet othermodifications could be made without deviating from the spirit and scopeof the claims.

1. (canceled)
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 7. A method for testing a sample for the presence of a firstligand, comprising: a) combining the sample with depletion molecules toobtain a mixture, said depletion molecules comprising molecules chosento specifically bind to analyte having a second ligand different frombut related to said first ligand to which said depletion molecules willnot bind, thereby generating bound second ligand and a remainder; b)applying at least the remainder to an immunoassay device; and c)inspecting the immunnoassay device to determine the presence of thefirst ligand.
 8. A method according to claim 7, wherein: said depletionmolecules comprise a conjugate of said second ligand with a largerparticle.
 9. A method according to claim 8, wherein: said largerparticle is a colored latex.
 10. A method according to claim 8, furthercomprising: generating said conjugate by obtaining freeze dried latexconjugate and a diluent and mixing them together.
 11. A method accordingto claim 7, further comprising: filtering said mixture to substantiallyremove said depletion molecules and bound second ligand prior to saidapplying.
 12. A method according to claim 11, wherein: said immunoassaydevice has a first sorbent strip having a first location for receiving asolution and defining a first migration path, a marker conjugate locatedon or in said first migration path, said marker conjugate adapted tobind to said first ligand, and a second sorbent strip distinct from saidfirst sorbent strip and having a second location for receiving saidremainder and defining a second migration path, and a test site locatedon or in at least one of said first sorbent strip and said secondsorbent strip, said test site having an immobilized ligand-bindingmechanism for said first ligand, and said first and second sorbentstrips touching each other at the test site location, wherein saidsecond location is removed from said test site such that remainderapplied to said second location requires time to migrate to said testsite and does not immediately wet said test site.
 13. A method accordingto claim 11, wherein: said immunoassay device has a first sorbent striphaving a first location for receiving a solution and defining a firstmigration path, a marker conjugate located on or in said first migrationpath, said marker conjugate adapted to bind to an interim binding agent,and a second sorbent strip distinct from said first sorbent strip andhaving a second location for receiving said remainder and defining asecond migration path, a second conjugate located on or in said secondmigration path, said second conjugate including a first-ligand-bindingmolecule and said interim binding agent, and a test site located on orin at least one of said first sorbent strip and said second sorbentstrip, said test site having an immobilized ligand-binding mechanism forsaid first ligand, and said first and second sorbent strips touchingeach other at the test site location, wherein said second location isremoved from said test site such that remainder applied to said secondlocation requires time to migrate to said test site and does notimmediately wet said test site.
 14. A method according to claim 13,wherein: said interim binding agent is biotin and said marker conjugatecomprises at least one of streptavidin and an anti-biotin antibody. 15.A method according to claim 11, wherein: said immunoassay device has afirst sorbent strip having a first location for receiving a solution anddefining a first migration path, a marker conjugate located on or insaid first migration path, said marker conjugate having a first interimbinding agent of a first pair adapted to bind to a second interimbinding agent of said first pair, and a second sorbent strip distinctfrom said first sorbent strip and having a second location for receivingsaid remainder and defining a second migration path, second moleculesand a second conjugate located on or in said second migration path, saidsecond molecules including a first-ligand-binding molecule and a firstinterim binding agent of a second pair, and said second conjugateincluding particles having bound thereto a second interim binding agentof said second pair and a second interim binding agent of said firstpair, and a test site located on or in at least one of said firstsorbent strip and said second sorbent strip, said test site having animmobilized ligand-binding mechanism for said first ligand, and saidfirst and second sorbent strips touching each other at the test sitelocation, wherein said second location is removed from said test sitesuch that remainder applied to said second location requires time tomigrate to said test site and does not immediately wet said test site.16. A method according to claim 15, wherein: said first interim bindingagent of said second pair is biotin, said second interim binding agentof said second pair is streptavidin, said first interim binding agent ofsaid first pair is anti-FITC antibody, and said second interim bindingagent of said first pair is FITC antigen.
 17. A method according toclaim 16, wherein: said particles of said second conjugate comprisewhite latex particles.
 18. A method according to claim 17, wherein: saidfirst ligand is HIV p24 ligand, and said first-ligand-binding moleculeof said second molecules are HIV p24 molecules.
 19. (canceled) 20.(canceled)
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